The production of red blood cells in mammals, erythropoiesis, is under the control of the hormone erythropoietin (EPO). EPO is normally present in low concentrations in plasma, where it is sufficient to maintain equilibrium between normal blood cell loss (i.e., through aging) and red blood cell production.
Anemia is a decrease in red blood cell mass caused by decreased production or increased destruction of red blood cells. EPO is currently used for treatment of the anemias associated with end-stage renal failure and acquired immunodeficiency syndrome (AIDS), particularly in subjects who are being treated with zidovudine (AZT). EPO is also used for amelioration of the anemia associated with cancer chemotherapy.
Another group of anemic disorders, each of which results from an inherited abnormality in globin production, is termed the hemoglobinopathies. Hemoglobinopathies include a spectrum of disorders that can be classified broadly into two types. The first type are those that result from an inherited structural alteration in one of the globin chains, for example sickle cell anemia. These disorders give rise to the production of abnormal hemoglobin molecules. The second major subdivision of hemoglobinopathies, the thalassemias, results from inherited defects in the rate of synthesis of one or more of the globin chains. This causes ineffective erythropoiesis, hemolysis, and varying degrees of anemia due to the inadequate production of red blood cells. Accordingly, EPO can be used in the treatment of anemias, for example, hemoglobinopathies which are characterized by low or defective red blood cell production and/or increased red blood cell destruction.
.beta.-thalassemia is a common single gene disorder arising from approximately 100 different mutations of the beta globin gene locus. Weatherall, D. J. (1994) in The Molecular Basis of Blood Diseases, Second Edition, Stamatoyannopoulos et al. eds., pages 157-205: WB Saunders, Philadelphia, Pa. The condition affects more than one million people worldwide. Current clinical management of severely anemic thalassemic patients generally involves transfusion with normal-matched donor blood, splenectomy, and other measures that help prolong circulating red blood cell survival. Schwartz et al. (1991) "The thalassemia syndromes," in Hematology: Basic Principals and Practice, First Edition, Hoffman et al. eds., pages 368-392, Churchill Livingstone, New York, N.Y.
Sickle cell anemia is a condition resulting from the production of abnormal .beta.-globin molecules due to an inherited mutation in the .beta.-globin gene. The mutation results in the substitution of valine for glutamic acid in the .beta.6 position of the molecule. Individuals homozygous for the abnormal .beta.-globin gene (.beta..sup.S) are symptomatic. Additionally, individuals heterozygous for .beta..sup.S and heterozygous for other abnormal .beta.-globin genes, for example, the .beta..sup.C gene or for .beta.-thalassemia, may also have symptoms of sickle cell anemia. Manifestations of the disease are due to the fact that, under certain physiologic conditions, the abnormal hemoglobin molecule (HbS) polymerizes, resulting in "sickling" of red blood cells. The consequences of sickling of red blood cells are increased cell destruction, resulting in anemia, and blockage in capillary beds which can result in damage to a number of organs such as the kidney, spleen, lung, bones and eyes.
Recent advances in molecular biology have led to an understanding of the molecular interactions between the various globin chains and, based on this knowledge, pharmacological treatment using butyrate, chotrimazole, or hydroxyurea (De Franceschi et al. (1996) Blood 87:1188-1195; Sauvage et al. (1993) Br. J. Haematol. 84:492) to increase globin concentration has been used in short-term clinical trials in small numbers of thalassemic patients to varying degrees of success. Olivieri et al. (1992) Blood 80:3258; Rachmilewitz et al. (1991) Blood 78:1145. The use of these pharmacological agents can mitigate the chain imbalance in beta-thalassemia and the precipitation of sickle hemoglobin in patients with sickle cell anemia. In a much smaller population of thalassemic patients, bone marrow transplantation of matched marrow has been shown to be curative (Thomas et al. (1982) Lancet 2:227-229; Lucarelli et al. (1990) N. Engl. J. Med. 322:417-421), however, this approach is only available to a limited number of patients. Since each of these approaches benefit only a few patients and carry the risk of substantial side effects, including death, novel therapeutic strategies are needed.
One new therapeutic approach for the treatment of thalassemia involves the delivery of high levels of systemic erythropoietin (EPO) to patients in order to increase red blood cell mass and survival. Administration of human EPO has been shown to transiently increase levels of erythropoieis and circulating red blood cell mass in rodents, normal primates, and in a small number of experimental studies with human patients. Al-Khatti et al. (1988) Trans. Assoc. Am. Physicians 101:54-61; Nagel et al. (1993) Blood 81:9-14; Olivieri et al. (1992) Blood 80:3258; Rachmilewitz et al. (1991) Blood 78:1145; Leroy-Viard et al. (1991) Blood 78:1596; Al-Khatti et al. (1987) N. Engl. J. Med. 317:415.
Administration of high doses of human EPO has shown efficacy in the treatment of sickle cell disease. For example, EPO has been shown to stimulate fetal hemoglobin formation in sickle cell anemia (Al-Khatti et al. (1988) Trans. Assoc. Am. Physicians 101:54-61), and some subjects with sickle cell disease who were treated with high doses of EPO responded with increased percentages of F-reticulocytes in peripheral blood (nagel et al. (1993) Blood 81:9-14). Further, high doses of EPO with adjunctive hydroxyurea has been shown to improve anemia in several sickle cell patients. Rodgers et al. (1993) New Engl. J. Med. 328:73.
Another new therapeutic strategy for the treatment of anemia entails gene therapy of hematopoietic stem cells, wherein transduced stem cells are used as a reservoir of gene-corrected cells. Williams et al. (1984) Nature 310:476-480; Lemischka et al. (1986) Cell 45:917-927. In particular, investigators have shown that murine bone marrow, transduced ex vivo by a retroviral vector containing the EPO gene, and then transplanted into lethally-irradiated thalassemic mice, can provide high levels of EPO secretion and improvement in the murine .beta.-thalassemic phenotype in a portion of the reconstituted animals. Villeval et al. (1994) Blood 84:928-933. However, several fundamental obstacles prevent gene therapy of hematopoietic stem cells from being realized. These obstacles include: unresolved problems with efficient delivery and integration into quiescent stem cells (Mulligan, R. C. (1993) Science 260:926-932); vector design and stability (Takekoshi et al. (1995) Proc. Natl. Acad. Sci. USA 92:3014-3018); transgene silencing (Challita et al. (1994) Proc. Natl. Acad. Sci. USA 91:2567-2571) and efficient engraftment and repopulation of gene-modified stem cells (Kohn et al. (1995) Nat. Med. 1:1017-1023).
Other researchers have reported the transduction of myoblast cells with DNA encoding EPO in vitro without using viral vectors, and the subsequent transplantation of the transduced cells into a murine host. International Patent Publication WO 95/13376, published May 18, 1995. Yet further research has explored ex vivo transduction of cells using adenovirus vectors. The transduced cells are then used to form a matrix (an "organoid") which is surgically implanted in the peritoneal cavity to secrete EPO. Naffakh et al. (1995) Proc. Natl. Acad. Sci. USA 92:3194-3198; Descamps et al. (1995) Gene Ther. 2:411-417. However, these methods have failed to provide for adequate levels of expression of EPO, for sufficient duration, in treated subjects, and are thus impractical.
Accordingly, the sustained delivery of high levels of EPO would be desirable in the treatment of hemoglobinopathies such as thalassemia and sickle cell disease. Further, the use of gene delivery methods that target cells other than hematopoietic stem cells, and avoid the problems with prior methods, would also be desirable.
Several experimenters have demonstrated the ability to deliver genes to muscle cells with the subsequent systemic circulation of proteins encoded by the delivered genes. See, e.g., Wolff et al. (1990) Science 247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Barr and Leiden (1991) Science 254:1507-1509; Dhawan et al. (1991) Science 254:1509-1512; Wolff et al. (1992) Human Mol. Genet. 1:363-369; Eyal et al. (1993) Proc. Natl. Acad. Sci. USA 90:4523-4527; Davis et al. (1993) Hum. Gene Therapy 4:151-159.
Genes have been delivered to muscle by direct injection of plasmid DNA. Wolff et al. (1990) Science 247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Barr and Leiden (1991) Science 254:1507-1509. However, this mode of administration generally results in sustained but low levels of expression. Low, but sustained expression levels, may be effective in certain situations, such as for providing immunity, but are generally not desirable for phenotypic improvement in most therapeutic methods.
Viral based systems have also been used for gene delivery to muscle. For example, human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses have been considered well suited for gene transfer because they are easy to grow and manipulate, and they exhibit a broad host range in vivo and in vitro. Adenoviruses are able to infect quiescent as well as replicating target cells and persist extrachromosomally, rather than integrating into the host genome.
Despite these advantages, adenovirus vectors suffer from several drawbacks which make them ineffective for long term gene therapy. In particular, adenovirus vectors express viral proteins that may elicit an immune response which may decrease the life of the transduced cell. This immune reaction may preclude subsequent treatments because of humoral and/or T cell responses. Furthermore, the adult muscle cell may lack the receptor which recognizes adenovirus vectors, precluding efficient transduction of this cell type using such vectors. Thus, attempts to use adenoviral vectors for the delivery of genes to muscle cells has resulted in poor and/or transitory expression. See, e.g., Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Acsadi et al. (1994) Hum. Mol. Genetics 3:579-584; Acsadi et al. (1994) Gene Therapy 1:338-340; Dai et al. (1995) Proc. Natl. Acad. Sci. USA 92:1401-1405; Descamps et al. (1995) Gene Therapy 2:411-417; Gilgenkrantz et al. (1995) Hum. Gene Therapy 6:1265-1274.
Gene therapy methods based upon surgical transplantation of myoblasts has also been attempted. See, e.g., International Publication No. WO 95/13376; Dhawan et al. (1991) Science 254:1509-1512; Wolff et al. (1992) Human Mol. Genet. 1:363-369; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hamamori et al. (1994) Hum. Gene Therapy 5:1349-1356; Hamamori et al. (1995) J. Clin. Invest. 95:1808-1813; Blau and Springer (1995) New Eng. J. Med. 333:1204-1207; Leiden, J. M. (1995) New Eng. J. Med. 333:871-872; Mendell et al. (1995) New Eng. J. Med. 333:832-838; and Blau and Springer (1995) New Eng. J. Med. 333:1554-1556. However, such methods require substantial tissue culture manipulation and surgical expertise, and, at best, show inconclusive efficacy in clinical trials. Thus, a simple and effective method of gene delivery to muscle, resulting in long-term expression of the delivered gene, would be desirable.
Recombinant vectors derived from an adeno-associated virus (AAV) have been used for gene delivery. AAV is a helper-dependent DNA parvovirus which belongs to the genus Dependovirus. AAV requires infection with an unrelated helper virus, such as adenovirus, a herpesvirus or vaccinia, in order for a productive infection to occur. The helper virus supplies accessory functions that are necessary for most steps in AAV replication. In the absence of such infection, AAV establishes a latent state by insertion of its genome into a host cell chromosome. Subsequent infection by a helper virus rescues the integrated copy which can then replicate to produce infectious viral progeny. AAV has a wide host range and is able to replicate in cells from any species so long as there is also a successful infection of such cells with a suitable helper virus. Thus, for example, human AAV will replicate in canine cells coinfected with a canine adenovirus. AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. For a review of AAV, see, e.g., Berns and Bohenzky (1987) Advances in Virus Research (Academic Press, Inc.) 32:243-307.
The AAV genome is composed of a linear, single-stranded DNA molecule which contains approximately 4681 bases (Berns and Bohenzky, supra). The genome includes inverted terminal repeats (ITRs) at each end which function in cis as origins of DNA replication and as packaging signals for the virus. The internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV rep and cap regions, respectively. These regions code for the viral proteins involved in replication and packaging of the virion. For a detailed description of the AAV genome, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129.
The construction of recombinant AAV (rAAV) virions has been described. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Numbers WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.
Recombinant AAV virion production generally involves cotransfection of a producer cell with an AAV vector plasmid and a helper construct which provides AAV helper functions to complement functions missing from the AAV vector plasmid. In this manner, the producer cell is capable of expressing the AAV proteins necessary for AAV replication and packaging. The AAV vector plasmid will include the DNA of interest flanked by AAV ITRs which provide for AAV replication and packaging functions. AAV helper functions can be provided via an AAV helper plasmid that includes the AAV rep and/or cap coding regions but which lacks the AAV ITRs. Accordingly, the helper plasmid can neither replicate nor package itself. The producer cell is then infected with a helper virus to provide accessory functions, or with a vector which includes the necessary accessory functions. The helper virus transactivates the AAV promoters present on the helper plasmid that direct the transcription and translation of AAV rep and cap regions. Upon subsequent culture of the producer cells, recombinant AAV virions harboring the DNA of interest, are produced.
Recombinant AAV virions have been shown to exhibit tropism for respiratory epithelial cells (Flotte et al. (1992) Am. J. Respir. Cell Mol. Biol. 7:349-356; Flotte et al. (1993) J. Biol. Chem. 268:3781-3790; Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617) and neurons of the central nervous system (Kaplitt et al. (1994) Nature Genetics 8:148-154). These cell types are well-differentiated, slowly-dividing or postmitotic. Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617; Kaplitt et al. (1994) Nature Genetics 8:148-154. The ability of AAV vectors to transduce nonproliferating cells (Podsakoff et al. (1994) J. Virol. 68:5656-5666; Russell et al. (1994) Proc. Natl. Acad. Sci. USA 91:8915-8919; Flotte et al. (1994) Am. J. Respir. Cell Mol. Biol. 11:517-521) along with the attributes of being inherently defective and nonpathogenic, place AAV in a unique position among gene therapy viral vectors.
Despite these advantages, the use of recombinant AAV virions to deliver the EPO gene to muscle cells in vivo has not heretofore been disclosed, particularly in the context of treating anemia in mammalian subjects.